Staphylococcus aureus is a gram positive human pathogen that causes a wide range of infections ranging from skin and soft tissue infections to life threatening diseases like sepsis and pneumonia. S. aureus is well known for its ability to acquire and evolve resistance mechanisms towards antimicrobial agents and meanwhile there are resistances towards all antibiotics available for clinical practice emphasizing a great need fo novel approaches to combat these pathogens. Currently there are no vaccines or therapeutics available for prevention or treatment of diseases caused by S. aureus. S. aureus is the most prevalent cause of hospital acquired infections and in recent years its rate of spread through the community has also been rising to an alarming level. Pneumonia is one of the most severe and prominent complications of S. aureus infection leading with 50,000 cases per year in the US alone. S. aureus pneumonia has been traditionally ventilator associated but in recent years it has been recognized also as a major cause of community acquired pneumonia primarily in otherwise healthy children and young individuals. S. aureus produces a wide range of secreted toxins with immune evasion and immune modulatory functions. Among these alpha hemolysin is known to be one of the most potent toxins and studies from multiple groups including ours show that alpha- hemolysin (Hla) also known as alpha toxin (AT) seems to be a a key virulence factor for S. aureus pneumonia and skin infection when tested in animal models reflecting human diseases. Hla cannot be used as a vaccine in its wild type form due to its toxic effect. The main goal of this Phase I SBIR is to complete proof of concept studies on a novel Hla vaccine designed based on crystallographic structure. Two vaccine candidates have been designed that represent a structural domain at the N terminus of Hla involved in oligomerization: i) the N-terminal 62 amino acids of AT (AT-62aa) and ii) an extended construct including amino acids 1-62 and 223-236 denoted as AT-79aa. Preliminary data presented in this proposal indicate induction of neutralizing antibodies and protective efficacy of AT-62aa in a pneumonia model of S. aureus infection, while a previously described AT-50aa (which disrupts the domain structure) only afforded a slight extension of life but was not protective when used with Alhydrogel adjuvant (in contrast to previous reports using IFA). This proposal is outlined in three specific Aims: In Aim 1, we will identify the best vaccine candidate based on immunogenicity studies of His-tagged AT-62aa protein using three different adjuvants that are either in licensed products (alum) or in clinical trial for human use (CpG and IDC-1001) and identify the optimal adjuvant. In specific Aim 2, we will develop a preliminary procedure for production and purification of a tag-free protein for the vaccine construct selected in Aim 1. We will also characterize the protein by biophysical methods, and perform a bridging study to confirm its immunogenicity in vivo. In specific Aim 3, we will perform a full set of proof of concept studies to show the protective efficacy of the optimal vaccine candidate in four different mouse models of infection: pneumonia, subcutaneous abscess model representing SSTI, bacteremia/sepsis, and surgical wound infection. This proposal is a collaborative effort between IBT and Dr. Jean Lee's laboratory at Brigham Women Hospital. Upon completion of the Phase I we envision a Phase II SBIR focused on completing preclinical development leading to submission of an Investigational New Drug (IND) application to the Food and Drug Administration.